GEN Exclusives

More »

Feature Articles

More »
Apr 1, 2013 (Vol. 33, No. 7)

Development and Evolution of PCR

  • Click Image To Enlarge +
    Forensic scientist places DNA samples from a crime scene into a thermocycler. The use of polymerase chain reaction (PCR) technology not only transformed the ways basic research can be conducted but has also impacted applications such as clinical diagnostics and forensic analyses. [Philippe Psaila/Science Source]

    In the six decades since Watson and Crick identified the double helical structure of DNA, a series of elegant and powerful experiments has established the broad outlines of how genetic information is encoded, replicated, and expressed.

    In the 1980s, the development of the polymerase chain reaction (PCR) dramatically simplified access to genomic information, facilitating both basic research studies and a wide variety of applications ranging from clinical diagnostics to forensic analyses.

    PCR—the enzymatic amplification of a specific DNA fragment targeted by two oligonucleotide primers—is a surprisingly simple concept but a technology that has become increasingly powerful and broadly implemented. Starting with a DNA or RNA template, repeated cycles of denaturation, primer annealing, and polymerase-mediated primer extension generate an exponential accumulation of a specifc targeted fragment that can be analyzed by a variety of methods.

    It is now difficult to conceive of any nucleic acid based study that does not incorporate PCR in some form in some stage of the experimental protocol.

  • PCR Properties

    In the ’80s, researchers explored a variety of signal or probe amplification systems for research and diagnostic applications. In contrast, PCR, with its sequential cycles of targeted oligonucleotide primed synthesis, is a target-amplification system, capable of amplifying unknown genomic regions between the primer sites.

    While sequence information is necessary for primer design, some mismatches between primer and template, primarily those at the 5´ end of the primer, could be tolerated. As a result, primers based on a sequence from one species could be used to amplify the homologous gene from related species, or those from one gene in a multigene family could amplify related genes.

    In addition, valuable sequence information, such as restriction sites to facilitate cloning or, more recently, adaptor and sample barcode sequences for next-generation sequencing (NGS), could be introduced at the ends of the amplicon via tolerated mismatches at the 5´ end of the primer. Labels could also be introduced into the amplicon using primers labeled at the 5´ end, facilitating both gel electrophoresis and probe hybridization analyses.

    PCR could also be used to confer on any DNA fragment the capacity to be replicated by simply ligating primer sites onto the ends of the targeted fragment(s), a property currently used in the preparation of shotgun libraries for NGS.

    PCR is essentially agnostic with respect to analytic methods: gel electrophoresis, probe hybridization, Tm melting analysis, sequencing, and a variety of other techniques have all been successfully applied to the analysis of amplicons.

    PCR’s ability to generate high concentrations of a specific labeled DNA target created a paradigm shift in oligonucleotide probe hybridization analysis. Previously, target DNA had been immobilized on a substrate, denatured, and hybridized under stringent conditions (so that a single mismatched base-pair would destablilize the target-probe duplex) to a vast molar excess of labeled probe (the “dot blot”).

    With PCR, a panel of sequence-specific oligonucleotide probes could be immobilized and then hybridized to a labeled amplicon (“reverse dot blot”). This basic principle became the basis of the linear array, probe-coupled beads, and microarray analysis, in which oligonucleotide probes are immobilized (by synthesis or deposition) on a membrane, bead, or chip.

  • Evolution of PCR Technology

    The three decades since the first PCR papers were published have seen many improvements, refinements, and modifications that have dramatically increased its analytic capabilities. However, the most transformative has surely been the introduction of a thermostable DNA polymerase.

    In the first PCR experiments designed to amplify β-globin from human DNA, we used the Klenow fragment of E. coli DNA polymerase. This system required adding fresh enzyme after the heat denaturation step of every PCR cycle, a cumbersome procedure (automated, during its brief reign, by a Rube Goldberg–like machine, known affectionately as Mr. Cycle). Because the primers were annealed and extended at 37°C, nontarget regions were often amplified along with the target.

    Under these conditions, the first successful genomic PCRs resulted in dramatic amplification and enrichment of the targeted 110 bp fragment of β-globin, but only around 1% of the amplified DNA was the target.

  • Taq Polymerase

    The introduction of the first thermostable polymerase in PCR (Taq polymerase, isolated from Thermus aquaticus) eliminated the need to add enzyme during each cycle, allowing the development of simple thermal cycling instruments to automate PCR. By allowing primer annealing and extension at elevated temperatures, the use of a thermostable polymerase also greatly increased the specificity of target amplification.

    Since the introduction of Taq polymerase, many different polymerases from a variety of thermophilic bacteria and hyperthermophilic archaea have been isolated, characterized, and used in PCR. Some have enhanced 3´ exonuclease activity (proofreading) and, thus, increased fidelity. Others have the ability to use RNA as a template.

    Discovery of DNA polymerases that could synthesize a cDNA strand from an RNA template made possible PCR assays for gene expression as well as detection and characterization of RNA viruses. Other polymerases have been “engineered” for particular properties by mutating specific functional sites so that, for example, dideoxyribonucleotides or ribonucleotides can be incorporated during the primer extension step. Other engineered polymerases are more discriminating in the extension of primers mismatched at the 3´ end, increasing the specificity of “allele-specific” PCR.

    The specificity of PCR has been further enhanced by chemically modified polymerases (“hot start” PCR) that remain inactive at lower temperatures and only extend primers at elevated temperatures. An alternative method of ensuring that polymerases are inactive at lower temperatures is the use of “aptamers,” small oligonucleotides that have been selected in vitro to bind to and inhibit a specific polymerase but that unfold at higher temperatures, releasing an active polymerase.

    These specificity-enhanced polymerases—along with primers that have been chemically modified at the 3´ end to prevent extension at lower temperatures—have been critical in achieving the sensitivity and specificity required by many clinical diagnostic applications.


Add a comment

  • You must be signed in to perform this action.
    Click here to Login or Register for free.
    You will be taken back to your selected item after Login/Registration.

Related content

Jobs

GEN Jobs powered by HireLifeScience.com connects you directly to employers in pharma, biotech, and the life sciences. View 40 to 50 fresh job postings daily or search for employment opportunities including those in R&D, clinical research, QA/QC, biomanufacturing, and regulatory affairs.
 Searching...
More »

GEN Poll

More » Poll Results »

Stopping Research Fraud

What is the best approach to curbing scientific misconduct and outright fraud?